Expression system for actinomycete-origin cytochrome p-450 in escherichia coli

ABSTRACT

This invention relates to a system for the expression of cytochrome P-450 gene in host  Escherichia coli,  and provides  Escherichia coli  which contains actinomycete ferredoxin gene and also ferredoxin gene and ferredoxin reductase gene which are xenogenic to  Escherichia coli.  Thus, this invention is useful for the promotion of effective single oxygen atom insertional reaction of a substrate organic compound.

TECHNICAL FIELD

This invention relates to a system for the expression of actinomycetecytochrome P-450 genes in Escherichia coli.

BACKGROUND TECHNOLOGY

Cytochrome P-450 enzymes (hereinafter referred to simply as “P-450s”)which are encoded by cytochrome P-450 genes are a general name of agroup of protoheme-containing proteins whose reduced form shows Soretband around 450 nm when bound to carbon monoxide. P-450s are bound tomicrosome in tissue of various kinds of animal or plant, or in fungi oryeasts, or to inner membrane of mitochondrion in tissue of some kind ofanimals. In certain kinds of bacteria and fungi, P-450s exist in solublestate.

P-450s show various types of substrate-specificity. Some P-450s haveabnormally so wide substrate-specificity that they can react withvarious kinds of organic compounds as substrate. Some, on the otherhand, have considerably strict substrate-specificity, and react onlywith comparatively limited kinds of organic compounds. P-450s also showexcellent stereo- and/or regio-specificity to reaction site. With regardto concrete functions, P-450s are known to catalyze reactions such ashydroxylation, epoxidation, dealkylation and denitrification, ofxenobiotics in a cell which expresses said P-450s. For example, most ofdrugs which are administered to human are metabolized and inactivated inthe body by various or specific action, such as hydroxylation, of P-450.In some cases, on the contrary, pharmacological effects of the drugs areimproved, or subsidiary action is enhanced. P-450 is, therefore,medically very important from the viewpoint of the research ofmetabolism of medicine or the development of prodrugs.

Thus, P-450s which have drug-metabolizing functions for higher organismsincluding human have been studied from every angle for long years.Although these enzymes are obtained from microsome fractions of liver ofhigher organisms, it is difficult to purify these enzymes into singleisozyme. On this account, there has been developed a technology toexpress functionally a gene which encodes single isozyme in a host suchas Escherichia coli or yeast, and to thus conveniently investigate themetabolic role of the enzyme.

P-450s of higher organisms which have such drug-metabolizing functionsas mentioned above have never been successfully applied to materialproduction on industrial scale. P-450s of higher organisms, whenfunctionally expressed in a host such as Escherichia coli or yeast, showonly low productivity as compared with P-450s of bacteria, and alsocause various side reactions. For these reasons, P-450s of higherorganisms have been used only restrictively.

In the case of P-450 originated from microorganisms such as fungi andbacteria, on the other hand, some kinds of P-450s are known to serve forthe production of industrially useful materials. Some of such kinds ofP-450s have actually been utilized for industrial production of usefulmedicine. Typical example is the production of pravastatin, a medicineto remedy hyperlipidemia, by means of the hydroxylation at 6β-positionof compactin with Storeptomyces carhophigus, a species of actinomycete(Watanabe et al., Gene, 163 (1995) 81-85, Japanese Patent ApplicationLaid-Open (Kokai) Publication No. Hei 6 (1994)-70780). There has alsobeen put into practice a method by which to produce active vitamin D₃ bymeans of the hydroxylation at 1α- and 25-positions of vitamin D₃ withuse of Pseudonocardia autotrophica, a species of actinomycete.

Such microbial conversion of drugs with use of actinomycete cytochromeP-450 enzymes as mentioned above have been carried out with use ofculture liquids or cells of actinomycetes which express said enzymes.There have also been used culture liquids wherein genes which encodeactinomycete P-450s have been introduced into Storeptomyces lividans,that is also a species of actinomycete and that is suitable as a host,to express the enzymatic activity. The microbial conversion of substratecompounds with the actinomycete strains which have such genes asmentioned above takes considerable time for the cultivation of thestrains and for the conversion of substrate compounds into desiredproducts. Furthermore, some enzymes need consideration onexpression-inducible conditions under which to increase the amount ofenzyme expressed. Moreover, some species of actinomycetes which are tobe used for the conversion have, in themselves, a system formetabolizing or degrading substrate or desired product, which causes theformation of by-products or the reduction of substrate or desiredproduct, and thus decreases the productivity of desired product.

There is also a report of experiment wherein, after the example offunctionally expressing a gene which encodes single isozyme of theabove-mentioned higher organism-originated P-450s in a host ofmicroorganism such as Escherichia coli, CYP105D1 gene which is aStoreptomyces griseus originated cytochrome P-450 gene was functionallyexpressed in Escherichia coli (Taylor et al., Biochemical andBiophysical Research Communications (1999) 263: 838-842). It seems that,in this expression system, some suitable electron donor for the P-450 inperiplasm of Escherichia coli hydroxylates hydrocarbons in cooperationwith the P-450 (Kaderbhai et al, Applied and Environmental Microbiology,67 (2001) 2136-2138). Such a P-450 gene-expressing system has a meritthat Escherichia coli as a host needs less time for cultivation ascompared with actinomycetes or the like.

DISCLOSURE OF INVENTION

The above-mentioned system for the conversion of organic compounds withmicroorganisms which have P-450s is intended to be used, for instance,for the application to biocatalyst or for the research of drugmetabolism. In consideration of application to biocatalyst, inparticular, more efficient bioconversion would be demanded. In order toachieve the efficient screening of industrially important and desiredactinomycete P-450 enzymes, there would be needed a suitable genelibrary as an object of robotized enzyme-assaying operation or otherconvenient and rapid enzyme-assaying operation in high throughputscreening or the like. Concretely, there is demanded a library which hasactinomycete-originated different cytochrome P-450 genes (actinomycetecytochrome P-450-expression library) and wherein microorganism,preferably handy and quick-growing microorganism, is used as a host inwhich each constituent clone is capable of expression.

In order to attain the above-mentioned objective, it would be useful touse, as a host, Escherichia coli which, at least, needs comparativelyshort time for cultivation, and which is considered to have only a fewsystems for the metabolism or degradation of substrate compounds orproducts from said substrate compounds. It has been confirmed, however,that only introducing an actinomycete P-450 gene into a host Escherichiacoli and cultivating the same, as in the above-mentioned Taylor et alwherein Escherichia coli is used as a host, does not achieve thefunctional expression of most of various kinds of P-450 genes which areoriginated from other species of actinomycete (or, in other words,enzymatic activity of P-450 is not shown as expected). Thus, theinventors of the present invention have studied how to construct asystem which is capable of functionally expressingactinomycete-originated various kinds of P-450 genes surely and withhigh enzymatic activity. As a result, they have found out that, when acertain electron transport system originated from microorganism which isxenogenic to Escherichia coli introduced together with P-450 gene and isthen allowed to co-express, the P-450 gene originated from variousspecies of actinomycete is functionally expressed.

Based on such a finding as mentioned above, this invention provides asystem for the expression of actinomycete cytochrome P-450 genes in hostEscherichia coli, wherein said Escherichia coli supports a recombinantDNA molecule which comprises xenogenic microorganism-originatedferredoxin gene, ferredoxin reductase gene and said cytochrome P-450gene in operable state.

Such an expression system as explained above is capable of theexpression of such a gene as mentioned in the above Taylor et al. whichencodes actinomycete cytochrome P-450, and which is incapable ofconjugating native electron transport system of Escherichia coli. Inother words, the expression system of this invention achieves expectedenzymatic activity of P-450 whether P-450 gene may conjugate nativeelectron transport system of Escherichia coli or not. Thus, the term“functionally express” in this specification means that a gene ofinterest expresses protein, which is encoded by the gene, in an activeform.

In the following, this invention is explained in more detail.

Host Escherichia coli means certain kinds of Escherichia coli which areusable for the propagation of vectors such as plasmids and phages and ofinserted genes. Any species of host will do if only, in a recombinantDNA experiment with use of host-vector system, a vector with anexogenous DNA fragment is capable of replication after transformation.As a host for said host-vector system, Escherichia coli on the marketwould be conveniently utilized.

Actinomycete cytochrome P-450 genes in this invention include P-450genes originated from any genus of bacteria that belong to orderActinomycetales if only the bacteria have P-450 genes which serve toachieve the objective of this invention in some form or other (e.g. onchromosome or plasmid). Thus, cytochrome P-450 genes include all thatencodes protein mentioned above which has such activity as to catalyzesingle oxygen atom insertional reaction in accordance with thisinvention. Although not restrictive, examples of P-450 genes which areintended to be incorporated into the expression system of this inventioninclude those which have the above-mentioned function, at least a partof whose DNA sequences have been determined, and each of whose sequenceinformation is available from gene data base (EMBL and GenBank),concretely, those which are originated from the following species ofactinomycte and which encode protein having the above-mentionedactivity, or those which have functions of cytochrome P-450 as mentionedbelow. Actinomycete Function of cytochrome P-450 Amycolatopsisorientalis Unknown Actinomadura verrucosospora Biosynthesis ofvercopeptin Amycolata autotrophica Unknown Amycolatopsis mediterraneiBiosynthesis of rifamycin Amycolatopsis mediterranei Biosynthesis ofbalhimycin Kitasatospora griseospola Biosynthesis of terpentecinMicromonospora griseorubida Biosynthesis of mycinamicin Micromonosporainyoensis Unknown Microtetraspora recticatena Hydroxylation of compactinMycobacterium smegmatis mc2155 Degradation of piperidine and pyrrolidineMycobacterium sp. FM10 Unknown Mycobacterium tuberculosis H37Rv 22 P-450genes in whole genome (function unknown) Myxococcus xanthus Polyketideantibiotic TA Pseudonocardia autotrophica Hydroxylation of vitamin D₃(old name: Amycolata autotrophica) Rhodococcus erythropolis Degradationof thiocarbamate herbicide Rhodococcus fascians (D188) Synthesis ofphytophysiologically active substance Rhodococcus ruber Degradation ofethyl-tert-butyl ether Saccharopolyspora erythraea Hydroxylation oferythromycin Streptoalloteichus hindustanus Unknown Streptomycesacidiscabies Biosynthesis of thaxtomin A Streptomyces albus UnknownStreptomyces ansochromogenes Biosynthesis of nikkomycin Streptomycesantibioticus Biosynthesis of oleandomycin Streptomyces antibioticusBiosynthesis of simocyclinone Streptomyces aureofaciens Ren71 UnknownStreptomyces avermitilis Formation of furan ring of avermectinStreptomyces avermitilis Biosynthesis of oligomycin Streptomycesavermitilis Biosynthesis of polyketide-4 Streptomyces avermitilisBiosynthesis of polyketide-9 Streptomyces avermitilis Biosynthesis ofother type polyketide Streptomyces avermitilis Biosynthesis of polyenemacrolide Streptomyces avermitilis Biosynthesis of peptide-7Streptomyces carbophilus Hydroxylation of compactin Streptomycesclavuligerus Biosynthesis of clavulanic acid Streptomyces coelicolorA3(2) 18 P-450 genes in whole genome (function unknown) Streptomycesfluvus Hydroxylation of compactin Streptomyces fradiae Biosynthesis oftylosin Streptomyces glaucescens Unknown Streptomyces griseolusDegradation of sulfonylurea herbicide Streptomyces griseus UnknownStreptomyces hygroscopicus Biosynthesis of rapamycin Streptomyceshygroscopicus Biosynthesis of FK520 var. ascomyceticus Streptomyceslavendulae Biosynthesis of mitomycin Streptomyces lavendulaeBiosynthesis of complestatin Streptomyces lividans Unknown Streptomycesmaritimus Biosynthesis of enterocin Streptomyces natalensis Biosynthesisof pimaricin Streptomyces nodosus Biosynthesis of amphotericinStreptomyces nogalater Biosynthesis of nogalamycin Streptomyces nourseiBiosynthesis of nystatin Streptomyces peucetius Hydroxylation ofdaunomycin Streptomyces peucetius Hydroxylation of daunomycin subsp.caesius Streptomyces rishiriensis Biosynthesis of coumermycin A1 strainDSM 40489 Streptomyces sclerotialus Unknown Streptomyces sp.Hydroxylation of FK-506 Streptomyces sp. Unknown Streptomyces spheroidsBiosynthesis of novobiocin Streptomyces tendae Biosynthesis ofnikkomycin Streptomyces thermotolerans Epoxidation of carbomycinStreptomyces venezuelae Biosynthesis of pikromycin, methymycin

The following actinomycete P-450 are also included as usable in thisinvention. Each of the following literatures gives guidance how toprepare gene which encodes each enzyme.

Compactin-hydroxylating enzyme originated from Streptomyces carbophilus(P-450_(sca)-2):

-   -   Watanabe et al., Gene 163 (1995) 81-85 or Japanese Laid-Open        (Kokai) Patent Publication No. Hei 6 (1994)-70780        Microtetraspora recticatena:    -   Japanese Laid-Open (Kokai) Patent Publication No. 2001-286293        Vitamin D3-hydroxylating enzyme originated from Amycolata sp.:    -   Sasaki et al., Applied Microbiology and Biotechnology (1992) 38:        152-157

Streptomyces roseochromogenes-originated progesterone-hydroxylatingenzyme (Berrie et al., Journal of Steroid Biochemistry & MolecularBiology 77 (2001) 87-96) can also be mentioned. Although gene sequenceof this Streptomyces roseochromogenes is not mentioned in publishedliteratures, function and biochemical properties of this P-450 enzymehave detailedly determined, and, on the basis of which information, itis easy to prepare gene which encodes said P-450 enzyme.

Cytochrome P-450-encoding genes (or P-450 genes) as mentioned in thisinvention include any gene so long as it can be isolated from total DNAsof the above-mentioned actinomycetes or can be amplified by PCRreaction, which is mentioned later, on the basis of information ofnucleotide sequences of said total DNAs, and so long as it is capable offunctional expression in the system of this invention for the expressionof P-450 genes. Also included in P-450 genes of this invention arepolynucleotides which are functionally equivalent to the above-mentionedgenes (also called native gene), and which have activity to catalyzesingle oxygen atom insertional reaction against corresponding substratesin the expression system of this invention. It is guessed thatcomplement base sequences of said equivalent polynucleotides hybridizewith corresponding native genes under a certain hybridization condition,e.g., under stringent condition in 2×SSC (standard saline citrate) at60° C., preferably in 0.5×SSC at 60° C., most desirably 0.2×SSC at 60°C. When each of the polynucleotides is lined up side by side with thecorresponding native gene, there would be shown homology of 80%,preferably 90%, most desirably at least about 95%. This “% homology”means percentage of nucleotide which is in common between two sequenceswhen the two sequences are lined up side by side with each other in anoptimum manner. [Thus, “% homology”=(number of coincidentpositions/total number of positions)×100. This can be calculated withuse of algorithm on the market. Such an algorithm is incorporated inNBLAST and XBLAST programs which are mentioned in Altschul et a., J.Mol. Biol. 215 (1990) 403-410.]

Ferredoxin gene which is incorporated in the expression system of thisinvention is a DNA molecule which is originated from microorganisms (orbacteria) which are xenogenic to host Escherichia coli. Ferredoxin genegenerally encodes protein which functions as an electron transporterhaving a molecular weight of about 6,000 to 14,000. Ferredoxin gene maybe originated from any bacteria except Escherichia coli so long as thebacteria participate in the functional expression of P-450 gene whenco-expressed with the above-mentioned actinomycete P-450 gene andfurther with ferredoxin reductase gene which will be mentioned later.Concrete examples of said bacteria, although not restrictive, includeactinomycete which may or may not be the same as mentioned above fromwhich P-450 gene is originated.

Also usable is ferredoxin gene originated from bacteria, e.g., of genusPseudomonas, which belong to different genus from that of actinomycetefrom which P-450 gene is originated. Examples of such a ferredoxin geneinclude putidaredoxin gene (also called camB) as mentioned in Petersonet al., The Journal of Biological Chemistry, 265 (1990) 6066-6073.

When ferredoxin gene is originated from the same actinomycete from whichP-450 gene is originated, P-450 gene and ferredoxin gene may sometimesconstitute a gene cluster in which said P-450 gene and ferredoxin geneexist adjacent to each other on genomic DNA. In such a case, a DNAfragment which contains both of said genes may be used in the expressionsystem of this invention. In the expression system of this invention,ferredoxin gene may exist with another ferredoxin. A preferable exampleof such a case is the use of ferredoxin gene originated fromactinomycete in combination with the above-mentioned camB originatedfrom Pseudomonas putida. Such a ferredoxin gene also includesfunctionally equivalent polynucleotide which can be specified in thesame manner as in the above-mentioned P-450 gene.

Ferredoxin reductase gene which is incorporated in the expression systemof this invention as an essential factor may be originated from bacteriawhich are xenogenic to host Escherichia coli, and which, undercircumstances, may also be xenogenic to the origin of P-450 gene.Concretely, ferredoxin reductase gene originated from any bacteria isusable in this invention so long as the bacteria are capable ofco-expression with the above-mentioned P-450 gene and with ferredoxingene, and so long as the gene encodes ferredoxin reductase which showsthe expected activity of P-450 enzyme, i.e., the product of said geneexpression of P-450, or, in other words, which catalyzes single oxygenatom insertional reaction against substrate. Examples of such ferredoxinreductase gene, although non-restrictive, include ferredoxin reductasegene originated from Streptomyces coelicolor (hereinafter sometimesreferred to as “fdr-1” or “fdr-2”) and putidaredoxin reductase geneoriginated from the above-mentioned Pseudomonas putida (hereinafterreferred to also as camA). Such a gene also includes functionallyequivalent polynucleotide which can be specified in the same manner asin the above-mentioned P-450 gene.

In the expression system of this invention, the above-mentioned P-450gene, ferredoxin gene and ferredoxin reductase gene are introduced inEscherichia coli an operable state. The term “operable state” means thatsaid genes are present in host in such a manner that all of the genesare capable of functional expression. In a typical example of such astate, all of the above-mentioned genes exist in a plasmid, which iscapable of autonomous replication in Escherichia coli, together withautonomously replicating sequence, promoter sequence, terminatorsequence and drug resistant gene, in a suitable order. Otherwise, all ofsaid genes exist in chromosome of host Escherichia coli such a mannerthat they are capable of functional expression via a chromosomal DNAintegrative vector. The above-mentioned P-450 gene, ferredoxin gene andferredoxin reductase gene may be arranged in any order in said plasmid.Usually, however, P-450 gene is preferably placed uppermost in thestream. When, in particular, P-450 gene and ferredoxin gene are used asa gene cluster fragment of the same origin, the following orders may bepreferable: P-450 gene-ferredoxin gene-ferredoxin reductase gene; P-450gene-ferredoxin gene-putidaredoxin reductase gene-putidaredoxin gene; orP-450 gene-putidaredoxin reductase gene-putidaredoxin gene.

Plasmid or vector which is usable in the above-mentioned expressionsystem may be capable of stable autonomous replication in Escherichiacoli, or may be an integrative vector which is capable of integratingchromosome of Escherichia coli with exogenous gene. Both can beavailable from those on the market, or by modification where necessary.Plasmids which have a strong promoter for gene transcription areconveniently used for the above-mentioned purpose. Examples of suchplasmids include those on the market, such as pET11 and pUC18.

Thus provided actinomycete P-450 gene expression system of thisinvention is usable for the screening of P-450 enzymes which aresuitable for the modification of various kinds of drugs or thebioconversion from precursor into desired drugs, or further for themanufacture of desired drugs from precursor.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1 shows the structure of plasmid pMoxAB.

FIG. 2 shows the structure of plasmid pMoxAB-fdr1.

FIG. 3 shows the structure of plasmid pMoxAB-fdr2.

FIG. 4 shows the structure of plasmid pMoxAB-camAB.

FIG. 5 shows the structure of plasmid pT7NS-camAB.

FIG. 6 shows the structure of plasmid pCBM-camAB.

FIG. 7 shows the structure of plasmid pSC154A1-camAB.

FIG. 8 shows the structure of plasmid pDoxA1-camAB.

In the following, this invention is concretely explained with workingexamples.

BEST MODE FOR WORKING THIS INVENTION

This invention will be further explained with reference to examples ofthe construction of P-450 gene which forms pravastatin of the followingformula:

or a mixture (which is called “RT-5.8 substances” in this specification)of isomers thereof which have the following formulae:

by means of single oxygen atom insertional reaction of compactin of thefollowing formula:

(or also existent as δ-lactone compound corresponding to the aboveformula) or a salt thereof.

Incidentally, pravastatin sodium is a clinically important medicine asan agent to cure hyperlipidemia.

Actinomycete which has an enzymatic activity to hydroxylate compactin(also called mevastatin) belongs to genus Streptomyces (JapaneseLaid-Open (Kokai) Patent Publication Sho 57 (1982)-50894, JapanesePatent No.2672551) or to genus Microtetraspora. As for the latter, whichhas been confirmed by the inventors of this invention, a DNA fragmentwhich contains P-450 gene can be prepared by the following process.

Preparation of P-450 Gene from Microtetraspora recticatena IFO 14525

Said gene can be obtained by polymerase chain reaction (PCR) with use ofprimers which have been designed in accordance with an amino acidsequence of the region which is known to keep amino acid sequence with ahigh probability among a family of lot of P-450 hydroxylation enzymes(J. Bacteriol. 172, 3335-3345 (1990)). For example, IFO 14525 strain iscultivated under certain cultivation conditions, and, then, thusobtained cells are crushed to give chromosomal DNA. Thus obtainedchromosomal DNA is subjected to PCR reaction with use of primers whichhave been designed from amino acid sequences for oxygen-binding domainand heme-binding domain which exist in common with P-450 hydroxylationenzyme family. There is obtained a DNA fragment which has been amplifiedby the PCR reaction, on the basis of which a further PCR reaction isconducted, and, thus, there is obtained flanking regions of the DNAfragment which has been amplified by the first PCR reaction (in thedownstream, there existed a gene which encoded ferredoxin). All of theabove-mentioned manipulation can be carried out by any method that isknown well in this art. Details of these sets of manipulation arementioned in the specification of Japanese Patent Application No.2001-47664 by the same applicant as that of the present application (thecontents of said specification are incorporated into the presentspecification by citation). Sequence No. 1 in the sequence listing showsa nucleotide sequence (and amino acid sequences encoded) which includesadjacent region of thus obtained P-450 gene.

In the above-mentioned sequence, a continuous nucleotide sequence frombase 313 to base 1533 corresponds to P-450 gene (moxA), and a continuousnucleotide sequence from base 1547 to base 1741 corresponds toferredoxin gene (moxB).

Preparation of P-450 Gene from Streptomyces sp. TM-6 or TM-7

From among a lot of microorganisms that belong to Streptomyces whichwere isolated from the soil in Japan, the applicant has identified theabove-captioned TM-6 and TM-7 strains as microorganisms which arecapable of biologically converting compactin as a substrate intopravastatin. Said strains were deposited on Apr. 25, 2001, at theInternational Patent Organism Depository (IPOD) in the NationalInstitute of Advanced Industrial Science and Technology (AIST) atTsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan, and thus havebeen received with Deposit Nos. FERM P-18311 and FERM P-18312.

Later, a demand was made on the above-mentioned IPOD, which is also aninternational depositary authority under the Budapest Treaty, for thetransfer of these strains TM-6 and TM-7 to said international depositaryauthority under said Treaty, and, thus, these strains have been receivedwith Deposit Nos. FERM BP-8002 and FERM BP-8003.

With regard to said TM-7 strain, a region of target gene was amplifiedby PCR in the same manner as in the above-mentioned IFO 14525, and,thus, the sequence of DNA fragment containing the target gene and itsadjacent region was determined. The result is shown in Sequence No. 2.In this sequence, a continuous nucleotide sequence from base 544 to base1758 corresponds to P-450 gene (boxA), and a continuous nucleotidesequence from base 1782 to base 1970 corresponds to ferredoxin gene(boxB). The manipulation to obtain these genes is mentioned detailedlyin the specification of Japanese Patent Application No. 2001-166412which has been filed by the applicant of the present application (thecontents of said specification are incorporated into the presentspecification by citation).

When compared with the sequence of P-450 gene of Streptomycescarbophilus SANK 62585 strain (FERM BP- 1145) which is mentioned, forinstance, in Japanese Patent No. 2672551, the nucleotide sequence of theabove-mentioned boxA was found to have a homology of about 75%. With theabove-mentioned moxA, the boxA has a homology of about 46%. Withhydroxylation enzyme gene of Streptomyces lividans, the boxA has ahomology of about 75%. Said boxA has a homology of about 46% with a geneencoding pyridylhomothreonine monooxygenase which is an intermediate inthe course of biosynthesis of nikkomycin by Streptomyces tendae Tji 901strain.

On the basis of the above explanation or explanation in working examplesmentioned below or, furthermore, on the basis of techniques which areknown well in this art or of information in gene database, anyoneskilled in the art would be able to obtain various kind of P-450 genesby means of firstly screening actinomycetes which are known (from typeculture catalogue published by ATCC) with respect to bio-conversion ofsubstrates for single oxygen atom insertion, then identifying strainshaving expected enzumatic activity, and thus conducting PCR operation asmentioned above. Hence, actinomycete cytochrome P-450 genes as called inthis invention include not only known ones but also all that skilledpersons could obtain.

The preparation of ferredoxin gene and ferredoxin reductase gene whichare included in the expression system of this invention, and theconstruction of expression system by means of operable connectionbetween these genes and P-450 genes, could be achieved quite easily byanyone skilled in the art in the same manner as in the above-mentionedP-450 genes, or in accordance with methods as mentioned in literatures(Sambrook et al, Molecular Cloning, A Laboratory Manual, 3^(rd) edition(2001), Cold Spring Harbor Laboratory Press, N.Y.), and in the light ofthe methods in working examples as mentioned later.

Thus constructed expression system for P-450 genes is capable offunctionally expressing P-450 genes under conditions where Escherichiacoli is grown. When such an expression system is incubated under asuitable condition together with a substrate for enzyme as a product ofP-450 gene (or when transformant as an expression system is cultivated),there is obtained a product wherein single oxygen atom has been insertedin substrate.

Cultivation is usually conducted on a medium which can be a nutritiousmedium for Escherichia coli, and which has no adverse effects onbiological conversion of substrate. Such a medium is composed ofsuitable carbon source, nitrogen source, inorganic salt and naturalorganic nutriment. As said carbon source, there can be used glucose,fructose, glycerol, sorbitol and organic acids, either singly or incombination. The concentration of these carbon sources when used issuitably about 1 to 10%, not particularly limited. As said nitrogensource, there can be employed one or two from ammonia, urea, ammoniumsulfate, ammonium nitrate and ammonium acetate. As said inorganic salt,there can be used salts such as potassium dihydrogenphosphate,dipotassium hydrogenphosphate, magnesium sulfate, manganese sulfate andferrous sulfate. As organic nutrient which has growth promoting effectson microorganism used, there can be used peptone, meat extract, yeastextract, corn steep liquor and casamino acids. Furthremore, a smallamount of vitamins and nucleic acids may be included in medium.

In the expression system of this invention, high-titer P-450 enzymeswith expected activity can be obtained when P-450 enzymes are induced ata temperature of about 25° C. or less, preferably at 20 to 24° C., afterhost Escherichia coli has been cultivated at a temperature suitable forthe growth of Escherichia coli, e.g. at 28 to 40° C.

The following is a detailed explanation of an example of construction ofexpression system for moxA gene originated from Microtetrasporarecticatena IFO 14525 which encodes a compactin-hydroxylating enzyme asan instance of actinomycete cytochrome P-450 enzymes. This invention is,however, not restricted at all by this example.

Polymerase Chain Reaction (PCR):

In the following example, PCT is conducted under conditions as follows.

(1) Condition where genomic DNA is used as a template:

(Composition of reaction liquid) Sterilized purified water 15 μlTwice-concentrated GC buffer I (Takara Shuzo) 25 μl dNTP mixed solution(dATP, dGTP, dTTP, 8 μl dCTP each 2.5 mM) Forward primer (100 pmol/μl)0.5 μl Reverse primer (100 pmol/μl) 0.5 μl Genomic DNA (10 ng/μl) 0.5 μlLA Taq (5 units/μl Takara Shuzo) 0.5 μl(Temperature condition)94° C. 3 minutes(98° C. 20 seconds; 63° C. 30 seconds; 68° C. 2 minutes) 30 cycles72° C 5 minutes(2) Condition where plasmid DNA (pMoxAB-fdr1) is used as a template:

(Composition of reaction liquid) Sterilized purified water 15 μlTwice-concentrated GC buffer I (Takara Shuzo) 25 μl dNTP mixed solution(dATP, dGTP, dTTP, dCTP each 2.5 mM) 8 μl Mox-3F primer (100 pmol/μl)0.5 μl Mox-5R primer (100 pmol/μl) 0.5 μl Plasmid DNA (1 ng/μl) 0.5 μlLA Taq (5 units/μl Takara Shuzo) 0.5 μl(Temperature condition)94° C. 3 minutes(98° C. 20 seconds; 63° C. 30 seconds; 68° C. 2 minutes) 25 cycles72° C. 5 minutes

EXAMPLE 1 Construction of Plasmid

(1) pT7-fdr1

PCR was carried out with use of primer FDR1-1F(5′-GCCATATGACTAGTGCGCCTCACAGACTGGAACGGGAATCTCATG -3′) (see SequenceNo.3) and FDR1-2R (5′-GCGAATTCTGTCGGTCAGGCCTGGTCTCCCGTCGGCCG-3′) (seeSequence No. 4) by using, as a template, genomic DNA of Streptomycescoelicolor A3(2) [imparted by John Innes Institute (Norwich, UK)], and,thus, there was amplified a 1.3-kb fragment of gene (hereinafterreferred to as fdr-1; see Sequence No. 5) encoding a protein which hashomology with ferredoxin reductases. This fragment was treated withrestriction enzyme Nde I and Bam HI, and was then subjected toelectrophoresis in 0.8% agarose gel. After the electrophoresis was over,the fdr-1 gene fragment was recovered, with use of SUPREC-01 (TakaraShuzo), from a gel piece containing said gene fragment, which had beencut out from the gel, and was purified. Said fragment was ligated to NdeI site and Bam HI site of Escherichia coli plasmid vector pET11a(manufactured by Stratagene Co.) with use of T4 DNA ligase, and, then,Escherichia coli EiDH5α was transformed with the resultant DNA reactionliquid, and, thus, pT7-fdr1 was constructed.

(2) pT7-fdr2

Under the same condition, PCR was carried out with use of primer FDR2-3F(5 ′-CGACTAGTGACGAGGAGGCAGACAAATGGTCGACGCGGATCAG-3 ′) (see Sequence No.6) and FDR2-4R (5′-CGGGATCCGACAACTATGCGACGAGGCTTTCGAGGG-3′) (seeSequence No. 7) by using genomic DNA of the above-mentioned Streptomycescoelicolor A3(2), and, thus, there was amplified a 1.3-kb fragment ofgene (hereinafter referred to as fdr2 see Sequence No. 8), which isdifferent from fdr-1, encoding a protein which has homology withferredoxin reductases. This fragment was treated with restriction enzymeBam HI and Spe I, and was then subjected to electrophoresis in 0.8%agarose gel. After the electrophoresis was over, fdr-1 gene fragment wasrecovered, with use of SUPREC-01 (Takara Shuzo), from a gel piececontaining said gene fragment, which had been cut out from the gel, andwas purified. Apart from that, plasmid pT7-fdr1 was treated with Bam HIand Spe I, and was then subjected to electrophoresis in 0.8% agarosegel. After the electrophoresis was over, pET11a vector fragment wasrecovered, with use of SUPREC-01 (Takara Shuzo), from a gel piececontaining said vector fragment which had been cut out from the gel, andwas purified. Said vector fragment and the above-mentioned fdr-1 genefragment were ligated to each other with use of T4 DNA ligase, and,then, Escherichia coli DH5α was transformed, and, thus, pT7-fdr2 wasconstructed.

(3) pT7-camAB

PCR was carried out with use of primer PRR-1F(5′-GCCCCCCATATGAACGCAAACGACAACGTGGTCATC-3′) (see Sequence No. 9) andPRR-2R (5′-GCGGATCCTCAGGCACTACTCAGTTCAGCTTTGGC-3′) (see Sequence No. 10)by using, as a template, genomic DNA of Pseudomonas putida ATCC17453,and, thus, there was amplified a 1.65 kb fragment (camAB fragment; seeSequence No. 16) which contained putidaredoxin reductase gene (camA) andputidaredoxin gene (camB) which was just downstream of said camA. Thisfragment was treated with restriction enzyme Nde I and Bam HI, and wasthen subjected to electrophoresis in 0.8% agarose gel. After theelectrophoresis was over, camAB fragment was recovered, with use ofSUPREC-01 (Takara Shuzo), from a gel piece containing said fragment,which had been cut out from the gel, and was purified. Said fragment wasligated to Nde I site and Bam HI site of Escherichia coli plasmid vectorpET11a (manufactured by Stratagene Co.) with use of T4 DNA ligase, and,then, Escherichia coli DH5α was transformed, and, thus, pT7-camAB wasconstructed.

(4) pMoxAB

PCR was carried out with use of primer Mox-1F(5′-GCCCCCCATATGACGAAGAACGTCGCCGACGAACTG-3′) (see Sequence No. 11) andMox-12R (5 ′-GCAGATCTAGTGGCTTCAGGCGTCCCGCAGGATGG-3′) (see Sequence No.12) by using, as a template, genomic DNA of IFO14525 strain, and, thus,there was amplified a 1.4-kb fragment (moxAB fragment) which contained agene (moxA) encoding compactin-hydroxylation enzyme and ferredoxin gene(moxB) which was adjacent downstream thereto. This fragment was treatedwith restriction enzyme Nde I and Bgl II I, and was then subjected toelectrophoresis in 0.8% agarose gel. After the electrophoresis was over,moxAB fragment was recovered, with use of SUPREC-01 (Takara Shuzo), froma gel piece containing said fragment, which had been cut out from thegel, and was purified. Said fragment was ligated to Nde I site and BamHI site of the above-mentioned plasmid pET11a with use of T4 DNA ligase,and, then, Escherichia coli DH5α was transformed with resultant reactionliquid, and, thus, plasmid pMoxAB was constructed.

(5) pMoxAB-fdr1 and pMoxAB-fdr2

PCR was carried out with use of primer Mox-1F(5′-GCCCCCCATATGACGAAGAACGTCGCCGACGAACTG-3′) (see Sequence No. 11 asmentioned above) and Mox-2R (5′-CGACTAGTGGCTTCAGGCGTCCCGCAGGATGG-3′)(see Sequence No. 13) by using, as a template, genomic DNA of IFO14525strain, and, thus, there was amplified a 1.4-kb fragment (moxABfragment) which contained a gene (moxA) encoding compactin-hydroxylationenzyme and ferredoxin gene (moxB) which was adjacent downstream thereto.This fragment was treated with restriction enzyme Nde I and Spe I, andwas then subjected to electrophoresis in 0.8% agarose gel. After theelectrophoresis was over, moxAB fragment was recovered, with use ofSUPREC-01 (Takara Shuzo), from a gel piece containing said fragment,which had been cut out from the gel, and was purified. Said fragment wasligated to Nde I site and Spe I site of the above-mentioned plasmidpT7-fdr1 with use of T4 DNA ligase, and, then, Escherichia coli DH5α wastransformed with resultant reaction liquid, and, thus, plasmidpMoxAB-fdr1 was constructed. FIG. 2 shows the structure of thispMoxAB-fdr1.

The same inserted fragment was ligated to Nde I site and Spe I site ofanother plasmid pT7-fdr2 with use of T4 DNA ligase, and, then,Escherichia coli DH5α was transformed with resultant reaction liquid,and, thus, plasmid pMoxAB-fdr2 was constructed. FIG. 3 shows thestructure of this pMoxAB-fdr2.

(6) pMoxAB-camAB

PCR was carried out with use of primer Mox-3F(5′-GGAGATATACATATGACGAAGAAC-3′) (see Sequence No. 14) and Mox-5R (5′-GCCCCCCATATGACGCACTCCTAGTGGCTTCAGGCGTCCCG-3′) (see Sequence No. 15) byusing, as a template, DNA of pMoxAB-fdr1, and, thus, there was amplifieda 1.5-kb fragment which contained a gene encoding cytochrome P-450enzyme having compactin-hydroxylating activity and ferredoxin gene(moxAB) which was adjacent downstream thereto. This fragment was ligatedto Nde I site of the plasmid pT7-camAB with use of T4 DNA ligase, and,then, Escherichia coli DH5α was transformed with resultant reactionliquid, and, thus, plasmid pMoxAB-camAB was constructed. FIG. 4 showsthe structure of this pMoxAB-camAB.

EXAMPLE 2 Preparation of Recombinant Which has Actinomycete CytochromeP-450 Enzymatic Activity

Escherichia coli BL21(DE3) was transformed with three plasmids, i.e.,pMoxAB-fdr1, pMoxAB-fdr2 and pMoxAB-camAB, and, thus, transformantstrains corresponding to these plasmids were obtained. Single colony ofeach of these strains was seeded on 2 ml of LB medium, and was subjectedto shake culture at 28° C. for 16 hours at 220 rpm. Thus obtainedculture liquid in an amount of 200 μl was mixed with an equal amount(200 μl) of 40% glycerol (sterilized) to give a glycerol culture, whichwas preserved at −80° C. until used. On the other hand, with use ofpMoxAB and pET11a which was used as a vector, Escherichia coli BL21(DE3)was transformed, and, thus, transformant strains corresponding to theseplasmids were obtained. Said transformant strains were used as control.

EXAMPLE 3 Production of Pravastatin and its Hydroxylated Analogues fromCompactin

(1) Production Process with Use of Static Cells:

Glycerol culture of transformant strain of BL21(DE3) as obtained in theabove Example 2 in an amount of 10 μd was added to 2 ml of LB medium towhich 50 μg/ml (final concentration) of ampicillin had been added, andwas then subjected to shake culture at 28° C. for 16 hours at 220 rpm.The resultant culture liquid in an amount of 250 μl was added to 25 mlof NZCYM medium to which 50 μg/ml (final concentration) of ampicillinhad been added, and was then subjected to shake culture at 37° C. for2.5 hours. Then, 25 μl of 100 mM IPTG and 25 μl of 80 mg/ml5-aminolevulinic acid were added in this order, and the resultantmixture was subjected to shake culture at 18-28° C. (this temperature ishereinafter called as “induction temperature”) for 16 hours at 120 rpm.Cells were recovered by centrifugation from 10 ml of the resultantculture liquid, and were then washed once with conversion buffer-2 (50mM NaH₂PO₄, 1 mM EDTA, 0.2 mM DTT 10% glycerol, [pH 7.3]). Subsequently,the cells were suspended in 1 ml of said buffer to give a suspension ofstatic cells. To this suspension of static cells, there were addedcompactin sodium salt (final concentration 250 μg/ml) and NADPH (finalconcentration 1 mM), and the resultant mixture was incubated at 32° C.for 24-48 hours (this time is hereinafter referred to as “conversiontime”) under shaking condition (220 rpm). Later, to 100 μl of thusobtained reaction liquid, there was added 100 μl of acetonitrile, andthe resultant mixture was subjected to vortex for one minute at roomtemperature, and was then centrifuged for 10 minutes at 16,000 rpm withan Epfendorf centrifugator. So obtained supernatant was analyzed withHPLC, and, thus, there were detected pravastatin and other hydroxylatedanalogues which had been formed by the hydroxylation of substratecompactin. The following shows detailed condition for this HPLC.

Analytical Apparatus: Shimadzu C-4RA Chromatopac

Column: J′ sphere ODS-H80 (YMC, Inc.), 75 mm×4.6 mm I.D.

Mobile phase A: Ion-exchange water/acetic acid/triethylamine=998:1:1 B:Methanol/acetic acid/triethylamine=998:1:1

Gradient time program:

0 minute Mobile phase A/B=50:50

3.00 minute Mobile phase A/B=10:90

3.50 minute Mobile phase A/B=10:90

3.51 minute Mobile phase A/B=50:50

6.00 minute End Flow rate: 2.0 ml/minute Detection: UV 237 nm Injectioncontent: 10 μl Column temperature: 40° C. Analysis time: 6 minutesRetention time: compactin 4.2 minutes pravastatin 2.7 minutes RT-5.8substances 3.6 minutes(2) Production Process by Fed-Batch Method:

Glycerol culture of transformant strain of BL21(DE3) in an amount of 10μl was added to 2 ml of LB medium to which 50 μg/ml (finalconcentration) of ampicillin had been added, and was then subjected toshake culture at 28° C. for 16 hours at 220 rpm. The resultant cultureliquid in an amount of 250 μl was added to 25 ml of M9-plus medium (M9salt, 0.4% glucose, 0.5% casamino acids, 100 μg/ml thiamin, 20 μl/mlthymine, 0.1 mM CaCl₂, 1 mM MgCl₂) to which 50 μg/ml (finalconcentration) of ampicillin had been added, and was then subjected toshake culture at 37° C. for 2.5 hours. Then, 25 μl of 100 mM IPTG and 25μl of 80 mg/ml δ-aminolevulinic acid were added in this order, and theresultant mixture was subjected to shake culture at 22° C. for 16 hoursat 120 rpm. To the resultant culture liquid, there was added 2.5 ml ofconversion mixture (2.5 mg/ml compactin sodium salt, 1 mg/ml FeSO₄.7H₂O,10 mM NADPH, 50% glycerol), and, thus, cultivation was continued at 22°C. for 96 hours. The period of time which has passed after the additionof this conversion mixture is hereinafter referred to as “cultivationtime”. Then, to 100 μl of this culture liquid, there was added 100 μl ofacetonitrile, and the resultant mixture was subjected to vortex for oneminute at room temperature, and was then centrifuged for 10 minutes at16,000 rpm with an Epfendorf centrifugator. So obtained supernatant wasanalyzed with HPLC, and, thus, there were detected pravastatin and otherhydroxylated analogues which had been formed by the hydroxylation ofsubstrate compactin.

Table 1 shows results of the measurement of the amount of pravastatinand RT-5.8 substance produced by static cells as mentioned in Example3(1) with use of Escherichia Coli transformant strain, under the proteininduction condition of 18-28° C. and with a conversion time of 48 hours.TABLE 1 Induction temperature Hydroxylated 18° C. 22° C. 25° C. 28° C.compactin RT-5.8 RT-5.8 RT-5.8 RT-5.8 (μg/ml) Pravastatin substancesPravastatin substances Pravastatin substances Pravastatin substancesBL21(DE3)/ 0 0 0 0 0 0 0 0 pET11a BL21(DE3)/ 0 0.18 0 0 0 0 0 0.3 pMoxABBL21(DE3)/ 0.24 1.24 1.72 8.48 0.43 1.92 0 0.61 pMoxAB-fdr1 BL21(DE3)/0.25 1.31 2.15 10.21 0 0.84 0.35 1.65 pMoxAB-fdr2 BL21(DE3)/ 3.84 22.076.09 33.55 4.05 16.53 0.97 5.49 pMoxAB-camAB

Productivity was the highest when induction temperature was 22° C.,under which condition each of strains wherein Streptomyces coelicolorA3(2)-originated ferredoxin reductase (fdr-1 or fdr-2) had beenco-expressed accumulated, in medium, 1.7 to 2.1 μg/ml of pravastatin and8.4 to 10.2 μg/ml of RT-5.8 substances. A strain wherein camAB had beenexpressed showed much higher productivity; it accumulated, in medium,6.09 μg/ml of pravastatin and 33.55 μg/ml of RT-5.8 substance. In thecase where there was used, as control, vector alone (BL21(DE3)/pET11a)or a strain which contained no gene to encode ferredoxin reductase(BL21(DE3)/pMoxAB), there were hardly detected pravastatin and RT-5.8substances.

Table 2 shows results of test of productivity of pravastatin inFed-batch method as mentioned in Example 4 (2). TABLE 2 Cultivation timeHydroxylated 4 hours 24 hours 48 hours 96 hours compactin RT-5.8 RT-5.8RT-5.8 RT-5.8 (μg/ml) Pravastatin substances Pravastatin substancesPravastatin substances Pravastatin substances BL21(DE3)/ 0 0 0 0 0 0 0 0pET11a BL21(DE3)/ 0 0 0 0 0 0 0 0 pMoxAB BL21(DE3)/ 0 0 0 0 0 0.87 00.61 pMoxAB-fdr1 BL21(DE3)/ 0 0 0 0 0 0.29 0 0.28 pMoxAB-fdr2 BL21(DE3)/0 0 0.28 2.82 0.64 7.74 0.95 12.44 pMoxAB-camAB

In the results with cultivation time of 96 hours, strains whereinStreptomyces coelicolor A3(2)-originated ferredoxin reductase (fdr-1 orfdr-2) had been co-expressed accumulated, in medium, 0.28 to 0.61 μg/mlof RT-5.8 substances while accumulating no pravastatin. A strain whereincamAB had been expressed showed high productivity; it accumulated, inmedium, 0.95 μg/ml of pravastatin and 12.44 μg/ml of RT-5.8 substances.In the case where there was used, as control, vector alone(BL21(DE3)/pET11a) or a strain which contained no gene to encodeferredoxin reductase (BL21(DE3)/pMoxAB), pravastatin and RT-5.8substances were not detected.

In a strain wherein camAB had been co-expressed, i.e., in Escherichiacoli wherein pMoxAB-camAB had been introduced, moxB (ferredoxin gene)and camB (putidaredoxin gene) among thus introduced genes overlap witheach other in their function. In order to know which gene among theconstituent genes contained in said pMoxAB-camAB are indispensable forthe expression of activity, the inventors of this invention constructeda plasmid which lacked one or two of said constituent genes, andintroduced the plasmid into Escherichia Coli. Table 3 shows results ofproductivity of hydroxylated compactin with use of static cells of thusprepared strain, and with a conversion time of 24 hours. TABLE 3Hydroxylated compactin Constituent gene (μg/ml) (+: existent; −:non-existent) RT-5.8 moxA moxB camA camB Pravastatin substancesBL21(DE3)/ − − − − 0 0 pET11a BL21(DE3)/ + + − − 0 0 pMoxABBL21(DE3)/ + + + − 0.17 0.82 pMoxAB- camA BL21(DE3)/ + − + + 0 0 pMoxA-camAB BL21(DE3)/ + + + + 1.67 9.96 pMoxAB- camAB

The above shows that three kinds of gene of moxA, moxB and camA areessential for the expression of activity, and that the addition of camBachieves a remarkable increase in activity; the yield of hydroxylatedcompactin increased about 10 times.

EXAMPLE 5 Construction of Plasmid

(1) pT7NS-camAB

PCR was carried out under the following condition with use of primerPRR-1F (5′-GCCCCCCATATGAACGCAAACGACAACGTGGTCATC-3′) (see 15 Sequence No.9) and PRR-2R (5′-GCGGATCCTCAGGCACTACTCAGTTCAGCTTTGGC-3′) (see SequenceNo. 10) by using, as a template, genomic DNA of Pseudomonas putidaATCC17453.

(Composition of reaction liquid) Sterilized purified water  15 μlTwice-concentrated GC buffer I (Takara Shuzo)  25 μl dNTP mixed solution(dATP, dGTP, dTTP, dCTP each   8 μl 2.5 mM) PRR-1F primer (100 pmol/μl)0.5 μl PRR-2R primer (100 pmol/μl) 0.5 μl Pseudomonas putida ATCC 17453Genomic DNA (10 ng/μl) 0.5 μl LA Taq (5 units/μl Takara Shuzo) 0.5 μl(Temperature condition)95° C. 3 minutes(98° C. 20 seconds; 63° C. 30 seconds; 68° C. 2 minutes) 30 cycles72° C. 5 minutes

An amplified 1.5-kb fragment (camAB fragment) which contained ferredoxinreductase gene (camA) and putidaredoxin gene (camB just downstreamthereto was treated with restriction enzyme Nde I and Bam HI, and wasthen subjected to electrophoresis in 0.8% agarose gel. After theelectrophoresis was over, camAB fragment was recovered, with use ofSUPREC-01 (Takara Shuzo), from a gel piece containing said gene fragmentwhich had been cut out from the gel, and was purified. Said fragment wasligated to Nde I site and Bam HI site of Escherichia Coli plasmid vectorpET11a (manufactured by Stratagene Co.) with use of T4 DNA ligase, and,then, Escherichia Coli DH5α was transformed, and, thus, pT7-camAB wasconstructed. To Nde I site of said plasmid pT7-camAB, there was ligatedby T4 DNA ligase one molecule of linker which had been prepared by theannealing of two kinds of synthetic oligo DNAs SP-1(5′-TATGCGTCACTAGTCGGGAGTGCGTTA-3′) (see Sequence No. 17) and SP-2(5′-TATAACGCACTCCCGACTAGTGACGCA-3′) (see Sequence No. 18), with whichEscherichia coli DH5α was transformed, and, thus, plasmid pT7NS-camABwas constructed. FIG. 5 shows the structure of pT7NS-camAB.

(2) pCBM-camAB

PCR was carried out under the following condition with use of primerCB-4F (5′-GCCCCCCATATGACAGCTTTGAATCTGATG-3′) (see Sequence No. 19) andCB-5R (5′-GCACTAGTCAGAGACGGACCGGCAGAC-3′) (see Sequence No. 20) byusing, as a template, total DNA of Streptomyces thermotoleransATCC11416, and, thus, there was prepared 1.25 kb fragment of ORF-A(cytochrome P-450 gene which encodes enzyme to epoxidize 12- and13-positions of carbomycin B) (gene sequence of ORF-A and the functionof ORF-A are mentioned in Bioscience Biotechnology Biochemistry vol.59,582-588, 1995; the contents of this literature is incorporated into thepresent specification by this citation).

(Composition of reaction liquid) Sterilized purified water 61 μl 10times-concentrated buffer (Takara Shuzo) 10 μl 25 mMgCl2 10 μl dNTPmixed solution (dATP, dGTP, dTTP, dCTP each 16 μl 2.5 mM) CB-4F primer(100 pmol/μl) 0.5 μl  CB-5R primer (100 pmol/μl) 0.5 μl  Total DNA (100ng/μl) of Streptomyces  1 μl thermotolerans ATCC11416 LA Taq (5units/μl, Takara Shuzo)  1 μl(Temperature condition)95° C. 3 minutes(98° C. 20 seconds; 63° C. 30 seconds; 68° C. 2 minutes) 30 cycles72° C. 5 minutes

This gene fragment was digested with restriction enzyme Nde I and Spe I,and was then subjected to electrophoresis in 0.8% agarose gel. After theelectrophoresis was over, ORF-A fragment was recovered, with use ofSUPREC-01 (Takara Shuzo), from a gel piece containing said genefragment, which had been cut out from the gel, and was purified. Saidfragment was ligated to Nde I-Spe I site of pT7NS-camAB with use of T4DNA ligase, and, with the resultant reaction liquid, Escherichia ColiDH5α was transformed, and, thus, plasmid pCBM-camAB was constructed.FIG. 6 shows the structure of pCBM-camAB.

(3) pSC154A1-camAB

Total DNA of Streptomyces coelicolor A3(2) (imparted by John InnesInstitute, Norwich, UK was digested with restriction enzyme Bam HI andPst I to give a 100 μg/μl solution of TE (10 mM Tris-HCl [pH 8.0], 1 mMEDTA). PCR was carried out under the following condition by using thisDNA as a template with use of primer 154A1-1F(5′-GCCCCCCATATGGCGACCCAGCAGCCCGCCCTC-3′) (see Sequence No. 21) and154A1-2R (5′-GCACTAGTCAGCCGGCGTGCAGCAGGACCGG-3′) (see Sequence No. 22),and, thus, there was prepared 1.2 kb gene fragment which encodedCYP154A1 (DNA sequence of gene which encodes Streptomyces coelicolorA3(2)-originated CYP154A1 has been published in gene database, e.g., GenBank, under gene name of SCE6.21).

(Composition of reaction liquid) Sterilized purified water 61 μl 10times-concentrated buffer (Takara Shuzo) 10 μl 25 mMgCl2 10 μl dNTPmixed solution (dATP, dGTP, dTTP, dCTP each 16 μl 2.5 mM) 154A1-1Fprimer (100 pmol/μl) 0.5 μl  154A1-2R primer (100 pmol/μl) 0.5 μl  TotalDNA (100 ng/μl) of Streptomyces  1 μl coelicolor A3(2) digested with BamHI-PstI LA Taq (5 units/μl, Takara Shuzo)  1 μl(Temperature condition)95° C. 3 minutes(98° C. 20 seconds; 63° C. 30 seconds; 68° C. 2 minutes) 30 cycles72° C. 5 minutes

This gene fragment was digested with restriction enzyme Nde I and Spe I,and was then subjected to electrophoresis in 0.8% agarose gel. After theelectrophoresis was over, CYP154A1-encoding gene fragment was recovered,with use of SUPREC-01 (Takara Shuzo), from a gel piece containing saidgene fragment which had been cut out from the gel, and was purified.Said fragment was ligated to NdeI-SpeI site of pT7NS-camAB with use ofT4 DNA ligase, and, with the resultant reaction liquid, Escherichia ColiDH5α was transformed, and, thus, plasmid pSC154A1-camAB was constructed.FIG. 7 shows the structure of pSC154A1-camAB. ps (4) pDoxA1-camAB

PCR was carried out under the following condition with use of primerDoxA-1F (5′-GCCCCCCATATGGCCGTCGACCCGTTCGCGTG-3′) (see Sequence No. 23)and DoxA-2R (5′-GCACTAGTCAGCGCAGCCAGACGGGCAGTTC-3′) (see Sequence No.24) by using, as a template, total DNA of daunomycin-producing bacteriumStreptomyces peucetius ATCC 29050, and, thus, there was prepared 1.2-kbfragment of doxA (cytochrome P-450 gene which participates in thebiosynthesis of daunomycin). DNA sequence of the doxA gene is mentionedin Journal of Bacteriology, vol. 181, No. 1, 305-318, 1999 (the contentsof this literature is incorporated into the present specification bythis citation).

(Composition of reaction mixture) Sterilized purified water 61 μl 10times-concentrated buffer (Takara Shuzo) 10 μl 25 mMgCl2 10 μl dNTPmixed solution (dATP, dGTP, dTTP, dCTP each 2.5 mM) 16 μl DoxA-1F primer(100 pmol/μl) 0.5 μl  DoxA-2R primer (100 pmol/μl) 0.5 μl  Total DNA(100 ng/μl) of Streptomyces peuceticus ATCC 29050  1 μl LA Taq (5units/μl, Takara Shuzo)  1 μl(Temperature condition)95° C. 3 minutes(98° C. 20 seconds; 63° C. 30 seconds; 68° C. 2 minutes) 30 cycles72° C. 5 minutes

This gene fragment was digested with restriction enzyme Nde I and Spe I,and was then subjected to electrophoresis in 0.8% agarose gel. After theelectrophoresis was over, doxa fragment was recovered, with use ofSUPREC-01 (Takara Shuzo), from a gel piece containing said gene fragmentwhich had been cut out from the gel, and was purified. Said fragment wasligated to Nde I-Spe I site of pT7NS-camAB with use of T4 DNA ligase,and, with the resultant reaction liquid, Escherichia Coli DH5α wastransformed, and, thus, plasmid pDoxA1-camAB was constructed. FIG. 8shows the structure of pDoxA1-camAB.

EXAMPLE 6 Microbial Conversion with Use of Escherichia coli RecombinantWherein Cytochrome P-450 Had Been Expressed

(1) Production of carbomycin A

Glycerol culture, in an amount of 10 μl, of Escherichia coli BL21(DE3)strain which had been transformed with pCBM-camAB among the plasmids asobtained in the above-mentioned Example 4 was added to 2 ml of LB mediumto which 50 μg/ml (final concentration) of ampicillin had been added,and the resultant mixture was subjected to shake culture at 28° C. for16 hours at 220 rpm. Thus obtained culture liquid in an amount of 250 μlwas added to 25 ml of NZCYM medium to which 50 μg/ml of ampicillin hadbeen added, and the resultant mixture was subjected to shake culture at37° C. for 2.5 hours. Then, 25 μl of 100 mM IPTG and 25 μl of 80 mg/mlδ-aminolevulic acid were added in order, and the resultant mixture wassubjected to shake culture at 22° C. at 120 rpm for 16 hours. Cells wererecovered by centrifugation from the resultant culture liquid, and werethen washed once with conversion buffer-2 (50 mM NaH₂PO₄, 1 mM EDTA, 0.2mM DTT, 10% glycerol, [pH 7.3]). Subsequently, the cells were suspendedin 3 ml of said buffer to give a suspension of static cells. To 600 μlof this suspension of static cells, 3 μl of 100 mg/ml methanol solutionof carbomycin B was added, and the resultant mixture was incubated at28° C. for five hours with shaking (220 rpm). Later, to thus obtainedreaction liquid, there was added 100 μl of 50% K2HPO₄ (pH 8.5) and 100μl of ethyl acetate, and the resultant mixture was subjected to vortex,and then to centrifugation for five minutes at 16,000 rpm with anEpfendorf centrifugator. A TLC plate was spotted with 10 μl of soobtained ethyl acetate phase, and was then subjected to development witha developer (ethyl acetate: diethylamine=100:2). Subsequently this platewas sprayed with 10% sulfuric acid, and heated at 100° C. for 10minutes. Spots on which color had come out were analyzed for coloringintensity with a dual-wavelength chromatoscanner CS-930 (manufactured byShimadzu Seisakusho) at a wavelength of 600 nm, and, thus, there wasevaluated the amount of carbomycin A (RF value in TLC: 0.64) which hadbeen formed by the epoxidation of substrate carbomycin B (RF value inTLC: 0.71). As a result, it was confirmed that carbomycin A had beenformed with a yield of 90 μg/ml. Then, substrate conversion reaction wasconducted with use of a control strain BL21(DE3) (pET11a) under the samecondition as stated above. As a result of analysis, no formation ofcarbomycin A was detected.

(2) De-ethylation of 7-ethoxycoumarin

Glycerol culture, in an amount of 10 μl, of Escherichia Coli BL21(DE3)strain which had been transformed with pSC154A1-camAB among the plasmidsas obtained in the above-mentioned Example 5 was added to 2 ml of LBmedium to which 50 μg/ml (final concentration) of ampicillin had beenadded, and the resultant mixture was subjected to shake culture at 28°C. for 16 hours at 220 rpm. Thus obtained culture liquid in an amount of250 μl was added to 25 ml of NZCYM medium to which 50 μg/ml ofampicillin had been added, and the resultant mixture was subjected toshake culture at 37° C. for 2.5 hours. Then, 25 μl of 100 mM IPTG and 25μl of 80 mg/ml δ-aminolevulinic acid were added in order, and theresultant mixture was subjected to shake culture at 22° C. at 120 rpmfor 16 hours. Cells were recovered by centrifugation from the resultantculture liquid, and were then washed once with conversion buffer-2 (50mM NaH₂PO₄, 1 mM EDTA, 0.2 mM DTT, 10% glycerol, [pH 7.3]).Subsequently, the cells were suspended in 6 ml of said buffer to give asuspension of static cells. To 1 ml of this suspension of static cells,5 μl of 50 mM DMSO solution of 7-ethoxycoumarin was added, and theresultant mixture was incubated at 28° C. for five hours with shaking(220 rpm). Later, to thus obtained reaction liquid, there was added 200μl of ethyl acetate, and the resultant mixture was subjected to vortex,and then to centrifugation for five minutes at 16,000 rpm with anEpfendorf centrifugator. There was taken out 100 μl of so obtained ethylacetate phase, which was then evaporated to dryness in vacuum. Theresultant dried pellet was dissolved in 1 ml of 100 mM potassiumphosphate buffer (pH 7.4). Thus obtained solution was 80-times diluted,and was then measured for fluorescence (wavelength: 460 nm) with F-2000spectrophotofluorometer (manufactured by Hitachi Science Systems, Co.)at an excitation wave length of 380 nm for the purpose of evaluation ofthe amount of 7-hydroxycoumarin which had been formed by de-ethylationof substrate 7-ethoxycoumarin. As a result, fluorescence intensity was2770, and, thus, the formation of 7-hydroxycoumarin was confirmed. Then,substrate conversion reaction was conducted with use of a control strainBL21(DE3) (pET11a) under the same condition as stated above. As a resultof analysis, fluorescence intensity was three or less.

(3) Dehydrogenation of 13-dihydrodaunomycin

Glycerol culture, in an amount of 10 μl of Escherichia coli BL21(DE3)strain which had been transformed by pDoxA1-camAB among the plasmids asobtained in the above-mentioned Example 5 was added to 2 ml of LB mediumto which 50 μg/ml (final concentration) of ampicillin had been added,and the resultant mixture was subjected to shake culture at 28° C. for16 hours at 220 rpm. Thus obtained culture liquid in an amount of 250 μlwas added to 25 ml of NZCYM medium to which 50 μg/ml of ampicillin hadbeen added, and the resultant mixture was subjected to shake culture at37° C. for 2.5 hours. Then, 25 μl of 100 mM IPTG and 25 μl of 80 mg/mlδ-aminolevulinic acid were added in order, and the resultant mixture wassubjected to shake culture at 22° C. at 120 rpm for 24 hours. Cells wererecovered by centrifugation from the resultant culture liquid, and werethen washed once with conversion buffer-2 (50 mM NaH₂PO₄, 1 mM EDTA, 0.2mM DTT, 10% glycerol, [pH 7.3]). Subsequently, the cells were suspendedin 4 ml of said buffer to give a suspension of static cells. To 1 ml ofthis suspension of static cells, 10 μl of 10 mg/ml methanol solution of13-dihydrodaunomycin was added, and the resultant mixture was incubatedat 28° C. for 24 hours with shaking (220 rpm). Later, to 400 μl of thusobtained reaction liquid, there was added 1.2 ml of acetone, and theresultant mixture was subjected to vortex, and was then extracted with300 μl of chloroform. Thus obtained extract was evaporated to dryness invacuum, and was then dissolved in 500 μl of 0.3 M hydrochloric acid, andthus obtained solution was heated at 80° C. for 30 minutes. Thissolution was extracted with 100 μl of chloroform, and thus obtainedextract was evaporated to dryness in vacuum. The resultant dried pelletwas dissolved in 100 μl of methanol, and the resultant solution wassubjected to HPLC under the following condition, and, thus, there wasdetected daunomycin which had been formed by the dehydrogenation ofsubstrate 13-dihydrodaunomycin.

(Analytical condition of HPLC)

Analytical apparatus: Shimadzu LC10 Chromatopac (manufactured byShimadzu Seisakusho)

Column: ZORBAX TMS (5 μl) 4.6 mm×250 mm I.D.

Mobile phase: To a mixture of water/acetonitrile/methanol/phosphoricacid=540:290:170:2 (volume ratio), 1.0 g of sodium lauryl sulfate wasadded and dissolved, and, to the resultant mixture, 2N NaOH was addedfor the adjustment of pH to 3.6. Flow rate: 1.5 ml/minute Wavelength fordetection: 254 nm Injection content: 20 μl Column temperature: 40° C.Analysis time: 20 minutes Retention time: 13-dihydrodaunomycin 4.8minutes daunomycin 5.9 minutes

Analysis detected 3.7 μg/ml of daunomycin. Then, substrate conversionreaction was conducted with use of a control strain BL21(DE3) (pET11a)under the same condition as stated above. As a result of analysis, noformation of daunonycin was detected.

INDUSTRIAL APPLICABILITY

In accordance with this invention, single oxygen atom insertionalreaction of organic compound as a substrate can efficiently be conductedwith use of a recombinant which has been constructed by use ofactinomycete cytochrome P-450 gene and Escherichia Coli a host.

This invention also provides a gene library suitable as an objective ofhigh throughput screening or other simple and rapid enzymatic assayscreening for the screening of industrially important and desiredactinomycete P-450 enzymes.

1. A system for the expression of actinomycete cytochrome P-450 genes inhost Escherichia coli, wherein said Escherichia Coli supports arecombinant DNA molecule which comprises xenogenicmicroorganism-originated ferredoxin gene, ferredoxin reductase gene aswell as said cytochrome P-450 gene, in operable state.
 2. An expressionsystem of claim 1 wherein ferredoxin gene and ferredoxin reductase geneare independently originated from some strain of actinomycete.
 3. Anexpression system of claim 1 wherein ferredoxin gene is originated frommicroorganism selected from the group consisting of those belonging togenus Microtetraspora and those belonging to genus Pseudomonas.
 4. Anexpression system of claim 1 wherein ferredoxin reductase gene isoriginated from microorganism selected from the group consisting ofthose belonging to genus Streptomyces and those belonging to genusPseudomonas.
 5. An expression system of claim 1 wherein actinomycetecytochrome P-450 gene and ferredoxin gene are originated from one andthe same gene cluster of actinomycete.
 6. An expression system of claim1 wherein ferredoxin reductase gene is originated from Streptomycescoelicolor.
 7. An expression system of claim 1 wherein actinomycetecytochrome P-450 gene and ferredoxin gene are originated from one andthe same gene cluster of actinomycete, and wherein ferredoxin reductasegene is originated from Streptomyces coelicolor.
 8. An expression systemof claim 1 wherein ferredoxin gene and ferredoxin reductase gene arerespectively putidaredoxin gene (camB and putidaredoxin reductase gene(camA) which are each originated from Pseudomonas putida.
 9. Anexpression system of claim 1 in which actinomycete cytochrome P-450 geneand ferredoxin gene are originated from one and the same gene cluster ofactinomycete, and which further contains, as another ferredoxin gene,putidaredoxin gene (camB originated from Pseudomonas putida.
 10. Anexpression system of claim 1 in which actinomycete cytochrome P-450 geneand ferredoxin gene are originated from one and the same gene cluster ofactinomycete, in which ferredoxin reductase gene is putidaredoxinreductase gene (camA) originated from Pseudomonas putida, and whichfurther contains, as another ferredoxin gene, putidaredoxin gene (camB)originated from Pseudomonas putida.
 11. An expression system of claim 1wherein actinomycete cytochrome P-450 gene and ferredoxin gene arerespectively compactin-hydroxylating enzyme-encoding gene (moxA)originated from Microtetraspora recticatina and ferredoxin gene (moxB)which is adjacent downstream to moxA.
 12. An expression system of claim1 wherein actinomycete cytochrome P-450 gene and ferredoxin gene arerespectively compactin-hydroxylating enzyme-encoding gene (moxA)originated from Microtetraspora recticatina and ferredoxin gene (moxB)which is adjacent downstream to moxA, and wherein ferredoxin reductasegene is ferredoxin reductase gene fdr-1 or fdr-2 originated fromStreptomyces coelicolor.
 13. An expression system of claim 1 in whichactinomycete cytochrome P-450 gene and ferredoxin gene are respectivelycompactin-hydroxylating enzyme-encoding gene (moxA originated fromMicrotetraspora recticatina and ferredoxin gene (moxB) adjacentdownstream to moxA, and which further contains, as another ferredoxingene, putidaredoxin gene (camB) originated from Pseudomonas putida, andin which ferredoxin reductase gene is putidaredoxin reductase gene(camA) originated from Pseudomonas putida.
 14. An expression system ofclaim 1 in which the induction of expression of cytochrome P-450 gene isconveniently carried out at 20 to 24° C.
 15. An expression system ofclaim 1 wherein said cytochrome P-450 gene comprises polynucleotidewhich is selected from the group consisting of polynucleotide having acontinuous nucleotide sequence from base 313 to base 1533 in SequenceNo. 1 or functionally equivalent polynucleotide with homology of atleast 80% to said nucleotide sequence, and polynucleotide having acontinuous nucleotide sequence from base 544 to base 1758 in SequenceNo. 2 or functionally equivalent polynucleotide with homology of atleast 80% to said nucleotide sequence.
 16. An expression system of claim1 wherein said ferredoxin gene comprises polynucleotide which isselected from the group consisting of polynucleotide having a continuousnucleotide sequence from base 1547 to base 1741 in Sequence No. 1 orfunctionally equivalent polynucleotide with homology of at least 80% tosaid nucleotide sequence, and polynucleotide having a continuousnucleotide sequence from base 1782 to base 1970 in Sequence No. 2 orfunctionally equivalent polynucleotide with homology of at least 80% tosaid nucleotide sequence.
 17. An expression system of claim 1 whereinsaid ferredoxin reductase gene comprises polynucleotide which isselected from the group consisting of polynucleotide having a continuousnucleotide sequence from base 118 to base 1377 in Sequence No. 5 orfunctionally equivalent polynucleotide with homology of at least 80% tosaid nucleotide sequence, and polynucleotide having a continuousnucleotide sequence from base 34 to base 1296 in Sequence No. 8 orfunctionally equivalent polynucleotide with homology of at least 80% tosaid nucleotide sequence.
 18. An expression system of claim 1 whereinsaid ferredoxin gene comprises polynucleotide having a continuousnucleotide sequence from base 1439 to base 1759 in Sequence No. 16 orfunctionally equivalent polynucleotide with homology of at least 80% tosaid nucleotide sequence.
 19. An expression system of claim 1 whereinsaid ferredoxin reductase gene comprises polynucleotide having acontinuous nucleotide sequence from base 115 to base 1380 in SequenceNo. 16 or functionally equivalent polynucleotide with homology of atleast 80% to said nucleotide sequence.
 20. A method to introduce ahydroxyl group at 6β- position of compactin with use of the expressionsystem of claim 12 or 13.